CN111676199A

CN111676199A - CAR-T technology for presenting and activating HSV-1 type oncolytic virus and application thereof

Info

Publication number: CN111676199A
Application number: CN202010586837.4A
Authority: CN
Inventors: 张同存; 祝海川; 张子健; 周勇; 顾潮江
Original assignee: Wuhan Bio Raid Biotechnology Co ltd
Current assignee: Wuhan Bio Raid Biotechnology Co ltd
Priority date: 2020-06-24
Filing date: 2020-06-24
Publication date: 2020-09-18
Anticipated expiration: 2040-06-24
Also published as: CN111676199B

Abstract

The invention discloses a system capable of killing solid tumor cells, which comprises HSV-1 type oncolytic virus, a chimeric antigen receptor and an scFv-synNotch-Cre element; wherein, scFv is a single-chain antibody of a targeted tumor specific antigen, Notch is a core element of transmembrane and shearing sites of Notch, and Cre is recombinase based on Loxp sites; the system accurately presents and activates HSV-1 type oncolytic virus replication through CAR-T cells, plays a role in combined treatment of solid tumors, has no toxic or side effect, and has an excellent effect.

Description

CAR-T technology for presenting and activating HSV-1 type oncolytic virus and application thereof

Technical Field

The invention relates to a technology for combining CAR-T cells and oncolytic viruses and application thereof, in particular to a technology for presenting CAR-T cells specifically and activating HSV-1 type oncolytic viruses with switches accurately based on a synNotch-Cre system and application thereof.

Background

Chimeric antigen receptor T cell therapy (CAR-T Immunotherapy) therapy is an emerging adoptive Immunotherapy, a new strategy for the treatment of malignancies following surgery and chemotherapy, gaining popularity in the treatment of B-cell acute leukemia with a complete remission rate of up to 90%. In 2017, two CAR-T immunotherapeutic products kymeriah (CTL-019) and yescatta (axicabagene ciloleucel, KTE-C10) were sequentially approved by the FDA in the united states for treatment of B-cell acute lymphoblastic leukemia in relapsed or refractory (r/r) children and young adults and patients with refractory, relapsed adult large B-cell lymphoma, respectively.

The CAR-T cell therapy achieves good curative effect on the aspect of hematologic malignancy treatment, but has an obvious short board in the solid tumor treatment, the solid tumor has compact tumor tissues and more complex tumor microenvironment, so that CAR-T cells are difficult to infiltrate from blood to tumor focuses, the solid tumor cells often express PD-L1 antigen, and a small amount of CAR-T cells are difficult to survive and proliferate and have the capacity of killing tumor cells after infiltrating because regulatory T cells (Tregs), marrow-derived suppressive cells (MDSCs) and the like exist in the microenvironment.

It is exciting that oncolytic viral therapy is just an emerging immunotherapy for solid tumor treatment and can complement CAR-T cells more perfectly for their deficiency in solid tumor treatment. The first oncolytic virus product, taliomogene laherparvec (T-vec, Imlygic), was approved by the FDA in the us 10 months of 2015 for melanoma treatment of recurrent or unresectable lesions. The oncolytic virus is a virus which can be specifically replicated and cracked in a tumor cell with an over-activated proliferation pathway after gene editing and is replicated and inhibited in normal tissues and cells, has unique advantages in solid tumor treatment due to the capability of specifically lysing the tumor cell and the characteristic of adjacent cell propagation of the oncolytic virus, can effectively lyse the tumor cell, destroy the tumor microenvironment, release tumor-related antigens while lysing the tumor cell to activate in vivo immune response, and has perfect complementary killing effect on solid tumors with CAR-T therapy.

However, most oncolytic viruses are administrated in an intratumoral administration mode, intratumoral administration is difficult to implement when focus is hidden, such as in the interior of a skull or an organ, and intravenous administration is easy to be eliminated by the anti-tumor immune response of an organism and accumulated in a liver part, so that the ideal anti-tumor effect cannot be achieved, and therefore, the development of an oncolytic virus administration scheme and a CAR-T combined application scheme are still the tumor treatment problems which need to be solved urgently.

Disclosure of Invention

The invention aims to solve the defects of the prior art, and provides a system for killing solid tumor cells, a preparation method and application thereof, wherein the system plays a role in treating solid tumors in a combined manner by accurately presenting and activating HSV-1 type oncolytic virus replication through CAR-T cells, and has an excellent effect.

In order to achieve the purpose, the invention adopts the following technical scheme.

The invention provides a system for killing solid tumors, which comprises HSV-1 type oncolytic virus, Chimeric Antigen Receptor (CAR) and scFv-synNotch-Cre element.

Preferably, the HSV-1 type oncolytic virus can be obtained by transforming multiple HSV-1 strains, and preferably one or more of the following strains: HSV-1JS1, HSV-1F, HSV-117, HSV-1E19, HSV-1KOS, HSV-1v23, HSV-1v29 and other clinical wild strains such as HSV1-2006-50683, HSV1-2009-20371, HSV1-2011-3153 and the like; more preferably HSV-117.

Further, based on HSV-117 strain, double copies of ICP34.5 and single copies of ICP47 are deleted by means of homologous recombination, and a CMV-EGFP-polyA tag gene expression frame is inserted into an ICP34.5 genomic site to obtain the oncolytic virus oHSV-1-X which can be specifically replicated in tumor cells.

Furthermore, a CMV-Loxp-Stop-Loxp element is inserted into the ATG upstream of the translation initiation codon of ICP4 by utilizing a homologous recombination method on the basis of oHSV-1-X to regulate the transcription of the ICP4 gene, so that the oncolytic virus Switch-oHSV-1-X which has a Switch and can replicate only when the Switch is opened is obtained. The Stop element may be formed by connecting a CDS region of a gene and a transcription termination signal in sequence; preferably, the CDS region of the gene is a fluorescent protein gene, more preferably, the fluorescent protein gene is mCherry, and the amino acid sequence of the fluorescent protein gene is shown as SEQ ID NO. 8; preferably, the transcription termination signal is an SV40PolyA signal, and the nucleotide sequence of the transcription termination signal is shown as SEQ ID NO.9, and preferably, the Loxp nucleotide sequence is shown as SEQ ID NO. 10. The CMV-Loxp-Stop-Loxp element is specifically CMV-Loxp-mCherry-polyA-Loxp, and a CMV promoter, a forward Loxp, an mCherry CDS, an SV40polyA and a forward Loxp are sequentially spliced from a 5 'end to a 3' end.

Preferably, the scFv-synNotch-Cre element consists of a signal peptide, a single chain antibody (scFv), Flag, Notch core, Cre recombinase, spliced in sequence from N segment to C end, wherein the scFv of the single chain antibody can specifically recognize a specific protein on the surface of a solid tumor, and the scFv of the CAR can be the same or different from the scFv of the CAR. Preferably, the signal peptide is a CD8 alpha signal peptide, and more preferably, the amino acid sequence of the signal peptide is shown as SEQ ID NO. 1; the Flag amino acid sequence is shown as SEQ ID NO. 31; the Notch Core element is a Notch transmembrane and cleavage site Core element, and more preferably, the amino acid sequence of the Notch Core element is shown as SEQ ID NO. 2; the Cre recombinase is an enzyme which can specifically recognize the Loxp site and can perform a DNA recombination function, and more preferably, the amino acid sequence of the Cre recombinase is shown as SEQ ID NO. 3. The scFv-synNotch-Cre element is linked to the CAR via the P2A peptide; preferably, the amino acid sequence of the P2A peptide is shown in SEQ ID NO. 16.

Preferably, the Chimeric Antigen Receptor (CAR) is spliced with a signal peptide, a single-chain antibody scFv, Strep tag II, CD8hinge, CD28TM + ICD, 4-1BB, and CD3 zeta in sequence from N-terminal to C-terminal. Preferably, the signal peptide is a CD8 alpha signal peptide, and more preferably, the amino acid sequence of the signal peptide is shown as SEQ ID NO. 1; the single-chain antibody scFv can specifically recognize specific proteins on the surface of a solid tumor; preferably, the Strep tag II amino acid sequence is shown as SEQ ID NO. 30; preferably, the CD8hinge amino acid sequence is shown as SEQ ID NO. 4; preferably, the amino acid sequence of CD28TM + ICD is shown in SEQ ID NO. 5; preferably, the 4-1BB amino acid sequence is shown as SEQ ID NO. 6; preferably, the amino acid sequence of CD3 ζ is as shown in SEQ ID NO. 7.

Preferably, the single chain antibody scFv can target a solid tumor target, such as EGFRvIII, HER2, MUC1, GD2, IL13aII and/or PSCA, etc., preferably, EGFR vIII and/or HER 2. scFv composed of heavy chain variable region V_HLinker connected with polypeptide, light chain variable region V_LComposition is carried out; preferably, the EGFR vIII-scFv heavy chain variable region V_HThe amino acid sequence of the EGFR vIII-scFv light chain variable region V is shown as SEQ ID NO.11_LThe amino acid sequence of (A) is shown as SEQ ID NO. 12; preferably, the HER2-scFv heavy chain variable region V_HThe amino acid sequence of the HER2-scFv light chain variable region V is shown as SEQ ID NO.13_LThe amino acid sequence of (A) is shown as SEQ ID NO. 14; preferably, the Linker amino acid sequence of the polypeptide chain is shown in SEQ ID NO. 15.

The invention also provides a gene vector of the recombinant chimeric antigen receptor, which takes lentivirus, adenovirus, adeno-associated virus, retrovirus, transposon vector or non-viral transient expression vector as a framework and is inserted with the nucleotide sequence which is connected by the Chimeric Antigen Receptor (CAR) and the scFv-synNotch-Cre through P2A; preferably, the transposon vector pT2/HB is used as a backbone, and the nucleotide sequence described above, which is linked by the Chimeric Antigen Receptor (CAR) and the scFv-synNotch-Cre via P2A, is inserted.

The invention also provides an immune cell expressing the CAR and the scFv-synNotch-Cre, which is obtained by transfecting the immune cell with the expression vector having the nucleotide sequence of the chimeric antigen receptor and the scFv-synNotch-Cre, wherein the immune cell is selected from umbilical cord blood, peripheral blood or IPSC-derived T cells and NK cells, and is preferably peripheral blood-derived T cells; the single chain antibody ScFv in the Chimeric Antigen Receptor (CAR) and scFv-synNotch-Cre elements may be the same or different and bind to tumor surface specific antigens, preferably HER2 and EGFR vIII.

The invention also provides a method for preparing the system for killing the solid tumors, which comprises the following steps:

firstly, based on HSV-117 strain, deleting double-copy ICP34.5 and single-copy ICP47 in a homologous recombination mode, and inserting CMV-EGFP-polyA label gene expression frame into ICP34.5 genomic site to obtain oncolytic virus oHSV-1-X which can be specifically replicated in tumor cells; further carrying out gene modification on oHSV-1-X, replacing a promoter of the ICP4 gene with a universal CMV promoter, connecting a Loxp-Stop-Loxp interrupt CMV promoter behind the CMV promoter to start the transcription of the subsequent ICP4 gene, and marking as Switch-oHSV-1-X. The ICP4 gene is used as an essential gene for completing replication of HSV-1 virus early, and the termination of the transcriptional expression of the ICP4 gene is equivalent to the termination of replication of the HSV-1 virus. And (3) performing virus genome sequencing after multiple purifications, and finally obtaining the high-purity oncolytic virus Switch-oHSV-1-X through virus amplification and concentration.

Subsequently loading oncolytic virus Switch-oHSV-1-X into synNotch-Cre-CAR-T, when the cell is identified with target cells around solid tumor tissues and combined with killing, simultaneously activating the cutting action of a Notch system, cutting Cre recombinase from an scFv-synNotch-Cre element and incorporating into nucleus, completing the deletion recombination of the Switch-oHSV-1-X on the basis of Loxp locus in the nucleus, excising spacer gene and transcription termination sequence (Loxp-Stop-Loxp) positioned between CMV promoter and ICP4 gene, completing the transcription and subsequent expression of the CMV promoter to ICP4 gene, further activating the replication and release of the oncolytic virus Switch-oHSV-1-X, infecting adjacent tumor cells, thereby cracking the tumor cells and opening tumor microenvironment, releasing tumor-related antigens and recruiting more CAR-T cells to participate in anti-tumor immune reaction, a schematic representation of the approach to achieve precisely controllable combined treatment of solid tumors presented by CAR-T cells and activating oncolytic virus replication is shown in figure 1.

The invention has the beneficial effects that:

1. the invention provides a CAR-T cell with an scFv-synNotch-Cre system, wherein the cell carries the synNotch-Cre system released based on signal cleavage of target protein besides the normal killing function of the CAR-T, when the extracellular scFv of the synNotch-Cre system is combined with the target protein, the shearing function of a Notch element near a cell membrane is activated, Cre recombinase connected inside the cell membrane of the synNotch system is sheared from the cell membrane, and the subsequent nuclear entry and recombination effects are generated;

2. the invention also provides an oncolytic virus Switch-oHSV-1-X with Cre recombinase mediated opening replication function, wherein an expression regulation Switch is added in front of the ICP4 gene, when the Switch is not opened, the ICP4 does not express, the oncolytic virus cannot replicate and amplify, and the expression of ICP4 can be opened under the deletion recombination of Cre recombinase to activate the replication of the oncolytic virus;

3. the invention provides a scheme for targeting presentation and precise regulation and control activation of oncolytic virus through CAR-T, the scheme is characterized in that the oncolytic virus with a Cre starting switch is delivered through CAR-T with a synNotch-Cre system, a small amount of replication of the oncolytic virus in a CAR-T cell transportation process can be prevented from influencing the activity of CAR-T cells in the CAR-T presenting oncolytic virus process, when the CAR-T carries the oncolytic virus to tumor cells, extracellular scFv is combined with target protein and rapidly starts replication of intracellular oncolytic virus, and then infection is carried out on adjacent target cells to destroy a tumor microenvironment, release tumor-related antigens to mediate CAR-T cells and an autoimmune system to form an immune response for combined killing of solid tumors.

4. The invention verifies that synNotch-CAR-T cells accurately present and activate the killing capacity of the oncolytic virus Switch-oHSV-1-X replication to target cells through an in vitro tumor killing experiment. After the synNotch-Cre-CAR-T cell presents and activates Switch-oHSV-1-X replication, the killing effect on MCF-7 and U251-MG of target cells is obviously superior to that of the synNotch-Cre-CAR-T and the oncolytic virus Switch-oHSV-1-X alone on the target cells. Overcomes the defect that the effect of the simple CAR-T cell therapy on solid tumors is not ideal, and overcomes the defects that the simple oncolytic virus administration therapy lacks targeting and easily causes in vivo immune response. The combination of the two can be used for targetedly treating solid tumors, and the effect of the treatment is far higher than that of oncolytic virus treatment or CAR-T treatment alone.

Drawings

FIG. 1 is a schematic representation of the synNotch-Cre system activating oncolytic virus Switch-oHSV-1-X;

FIG. 2 is a schematic representation of the DNA fragments of HER2-CAR and EGFR vIII-CAR;

FIG. 3 is a schematic representation of the HER2-synNotch-Cre and EGFR vIII-synNotch-Cre system DNA fragments;

FIG. 4 is a schematic representation of the HER2-synNotch-Cre-CAR and EGFR vIII-synNotch-Cre-CAR system DNA fragments;

FIG. 5 is a plasmid map of pT2-HER2-synNotch-Cre-CAR and pT2-EGFR vIII-synNotch-Cre-CAR;

FIG. 6 is a schematic diagram of the Switch-oHSV-1-X gene modification;

FIG. 7 shows the results of the measurements of the transfection efficiencies of HER2-synNotch-Cre-CAR-T and EGFR vIII-synNotch-Cre-CAR-T;

FIG. 8 is a synNotch-Cre-CAR-T cell ratio assay loaded with oncolytic virus Switch-oHSV-1-X;

FIG. 9 is a genomic copy number assay in which oncolytic virus Switch-oHSV-1-X is activated to initiate replication;

FIG. 10 is a graph of the specific presentation of Switch-oHSV-1-X to the corresponding target cells MCF-7 and U251-MG, respectively, following the load activation of HER2-synNotch-Cre-CAR-T and EGFR vIII-synNotch-Cre-CAR-T;

FIG. 11 shows the synNotch-Cre-CAR-T cell killing results for oncolytic virus Switch-oHSV-1-X: cell membrane (membrane), Target cell (Target cell), Antigen (Antigen), cleavage (cleavage), Nucleus (Nucleus), Recombination (Recombination), switch on (Swich on), Virus replication (Virus replication), effector cell: target cells (Effect: Target), tumoricidal efficiency (Specific Lysis), cell number (count), control (control), virus (virus), activation (activation), and virus particles (particles).

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: construction of PT2-HER2-synNotch-Cre-CAR and PT2-EGFRvIII-synNotch-Cre-CAR plasmids

1. Artificially synthesizing a sequence containing a single-chain antibody against HER2, namely splicing and synthesizing nucleotide fragments shown by a nucleotide sequence SEQ ID NO.17 of a CD8a signal peptide, a nucleotide sequence SEQ ID NO.18 of a heavy chain variable region, a nucleotide sequence SEQ ID NO.22 of a connecting polypeptide Linker and a nucleotide sequence SEQ ID NO.19 of a light chain variable region in sequence to form SP-HER 2-scFv. Similarly, a sequence of a single-chain antibody EGFRvIII is artificially synthesized, namely nucleotide fragments shown by a nucleotide sequence SEQ ID NO.17 of a signal peptide of CD8a, a nucleotide sequence SEQ ID NO.20 of a heavy chain variable region, a nucleotide sequence SEQ ID NO.22 of a Linker polypeptide and a nucleotide sequence SEQ ID NO.21 of a light chain variable region are sequentially spliced and synthesized to form the SP-EGFRvIII-scFv.

2. A human cDNA library is taken as a template, primers are designed for PCR to respectively amplify a fragment CD8hinge nucleotide sequence SEQ ID NO.23, a CD28TM + ICD nucleotide sequence SEQ ID NO.24, a 4-1BB nucleotide sequence SEQ ID NO.25 and a CD3 zeta nucleotide sequence SEQ ID NO.26, a Strep tag II nucleotide fragment SEQ ID NO.27 is obtained in a primer complementary mode, and SP-HER2-scFv, SP-EGFR vIII-scFv, fragments Strep tag II, CD8hinge, CD28TM + ICD, 4-1BB and CD3 zeta are sequentially amplified and connected into complete chimeric antigen receptor CAR structures HER2-CAR and EGFRvIII-CAR with Strep tag II by using an Overlap PCR technology, wherein the structure schematic diagram is shown in FIG. 2.

3. The nucleotide sequence SEQ ID No.28 of Notch core is amplified by using a rat cDNA library as a template and the nucleotide sequence SEQ ID No.29 of Cre recombinase is amplified by using a P1 phage genome as a template. The nucleotide sequence SEQ ID NO.32 of the Flag tag protein is obtained in a primer complementary mode. Similarly, SP-HER2-scFv and SP-EGFRvIII-scFv were amplified sequentially with Flag, Notch core and Cre recombinase to form Flag-tagged HER2-synNotch-Cre and EGFRvIII-synNotch-Cre, respectively, by the Overlap PCR technique, and the schematic structure is shown in FIG. 3. Then connecting HER2-CAR, P2A peptide and HER2-synNotch-Cre into HER2-synNotch-Cre-CAR in sequence by using an Overlap PCR technology; and sequentially connecting the EGFRvIII-CAR, the P2A peptide and the EGFRvIII-synNotch-Cre into the EGFRvIII-CAR-synNotch-Cre, wherein the structural schematic diagram is shown in FIG. 4.

4. Plasmid pT2/HB was double digested with HandIII and EcoRI restriction enzymes, the product was subjected to 0.8% agarose gel electrophoresis and gel-cut recovery in a 1.5mL centrifuge tube, the corresponding fragment was recovered using the agarose gel recovery kit from Axygen, and the purity and concentration of the product were determined.

5. Adding the pT2/HB vector recovered fragment and HER2-synNotch-Cre-CAR and EGFR vIII-synNotch-Cre-CAR into a 1.5mL centrifuge tube according to the molar ratio of 1:2, adding an Exnase II ligase (Vazyme) and a homologous recombinase 5 XCE II buffer, and reacting for 0.5 hour at 37 ℃; taking 10 mu L of the connecting liquid, adding 100 mu L of DH5 alpha competent cells, carrying out ice bath for 30min, then carrying out heat shock at 42 ℃ for 90s, adding 500 mu L of soc culture medium at 37 ℃ and 220rpm, and culturing for 2 hours; centrifuging at 4000g for 1min to remove about 400 μ L of supernatant, then gently blowing and uniformly mixing the thallus precipitate, coating the mixture on an LB flat plate, and culturing at 37 ℃ for 12 hours; single colonies were picked on the plate, inoculated into 5mL of LB liquid medium at 37 ℃ and 220rpm for 12 hours.

6. Plasmids are extracted by an Axygen miniprep kit to obtain plasmids pT2-HER2-synNotch-Cre-CAR and pT2-EGFR vIII-synNotch-Cre-CAR, and after the generation sequencing verification of the science and technology company of the engineering and biological engineering (Shanghai) corporation, strains are preserved. The plasmid map schematic of pT2-HER2-synNotch-Cre-CAR, pT2-EGFRvIII-synNotch-Cre-CAR are shown in FIG. 5.

Example 2 preparation and sequencing of plasmids pT2-HER2-synNotch-Cre-CAR and pT2-EGFRvIII-synNotch-Cre-CAR

1. Preparation of plasmids

DH 5. alpha. strain containing plasmids pT2-HER2-synNotch-Cre-CAR and pT2-EGFR vIII-synNotch-Cre-CAR was inoculated into 250mL LB medium containing 100. mu.g/mL ampicillin and cultured overnight at 37 ℃ and 220 rpm. The culture was centrifuged at 6000g for 20min at 4 ℃ and the supernatant was discarded.

Take out the Buffers P1 in EndoFree plasma mega kit (Qiagen), add 120mL of precooled Buffers P1 to the E.coli pellet obtained by centrifugation, cover the centrifuge cap, and vigorously shake the centrifuge flask to completely disperse the E.coli pellet in Buffers P1.

120mL of Buffers P2 was added to the flask, the flask was covered with a cap and placed on a roller mixer, the speed was slowly increased to 50rpm, and the mixture was thoroughly mixed and then left at room temperature for 5 min.

Adding 120mL of Buffers P3 into a centrifuge bottle, covering the centrifuge bottle with a bottle cap, placing the centrifuge bottle on a roller mixer, slowly increasing the speed to the maximum rotation speed of 70rpm of the roller mixer, and thoroughly mixing until the centrifuge bottle is white non-sticky and fluffy mixed liquid. Centrifuge at 9000g for 15min at 4 ℃.

50mL of Buffer FW was poured into the QIAfilter card, and the supernatant obtained by centrifugation was poured into the QIAfilter card, and gently stirred and mixed. And pumping and filtering the mixed solution into a corresponding marked glass bottle.

20mL Buffer ER was added to each glass vial, mixed 6 times upside down and incubated at-20 ℃ for 30 min.

The labeled mega columns were placed on corresponding racks, and 35mL of Buffers QBT was added to each mega column to equilibrate and drain by gravity.

And (3) pouring all the liquid in the glass bottles into the corresponding marked mega columns in batches, and adding 200mL of Buffer QC into each mega column in batches for washing after the liquid in the columns is drained. After the liquid in the column had run out, the waste liquid in the waste liquid collection tray was poured into a 50mL clean centrifuge tube.

40mL Buffer QN was added to each mega column, the effluent was collected using a 50mL clean centrifuge tube, mixed by inverting 6 times, and dispensed 20mL into another clean labeled 50mL centrifuge tube.

To each 50mL centrifuge tube, 14mL of isopropanol (room temperature) was added, and the mixture was mixed by inverting the mixture 6 times. Centrifuge at 15000g for 50min at 4 ℃.

The supernatant was aspirated off the clean bench, and 3.5mL of endo-free water was added to each tube to rinse without dispersing the bottom precipitate. Centrifuge at 15000g for 30min at 4 ℃. Buffer TE from an EndoFree plasma mega kit was placed in a 65 ℃ oven for preheating.

And (4) completely absorbing the centrifuged supernatant in the clean bench, and drying in the clean bench (volatilizing residual absolute ethyl alcohol for about 10 min).

Taking out the Buffer TE in the oven, adding 1mL of Buffer TE into each tube in a clean bench, blowing for 10 times by using a gun, and then putting the tube into the oven at 65 ℃, wherein the tube wall is uninterruptedly knocked to promote the precipitate to be completely dissolved. Centrifuging at 4 deg.C at 4000g for 1min to throw the liquid on the tube wall to the tube bottom, blowing, beating and mixing.

The liquid was transferred in its entirety to endotoxin-free, pyrogen-free and nuclease-free correspondingly labeled 1.5mL centrifuge tubes in a clean bench. Aspirate 2. mu.L and measure the plasmid concentration using a microspectrophotometer to obtain a large amount of pT2-HER2-synNotch-Cre-CAR plasmid and pT2-EGFR vIII-synNotch-Cre-CAR plasmid.

2. Sequencing of target genes

20 mu L (500ng) of plasmid DNA is respectively taken and sent out for sequencing, whether the target gene of a product produced by the plasmid is changed or not is checked according to an original seed sequence, and the target gene cannot be changed in the process of fermentation culture and amplification of working seeds under a stable process, so that the method can be used for production and correct expression of protein in the next link.

Example 3 preparation of HER2-synNotch-Cre-CAR-T, EGFR vIII-synNotch-Cre-CAR-T cells

1. T cell transfection:

collecting 100mL of peripheral blood of a healthy donor, separating mononuclear cells by adopting a Ficoll lymphocyte separation solution, counting, using a proper amount of CD3MicroBeads and human (Meitian and whirlpool) to sort CD3 positive cells, and using 1.0-2.0 × 10⁶cell/mL density in complete T cell culture (OpTsizer)^TMCTS^TMT-Cell Expansion Basal Medium，OpTmizer^TMCTS T-Cell Expansion Supplement (Invitrogen), IL-2 (double Lut pharmaceutical industry)) at 500IU/mL, and the culture was carried out at a rate of 10⁶mu.L of Dynabeads Human T-Activator CD3/CD28(Invitrogen) was added to each cell to activate the T cells.

After 24 hours, two portions of each 2 × 10⁶The supernatant was discarded by centrifuging each T cell at 300g for 5 minutes, adding 3mL of PBS solution and shakingCentrifuging at 300g for 5 minutes after uniform mixing to remove supernatant, repeating the PBS cleaning step, sucking the supernatant by using a suction head, adding 500 mu L of electrotransfer buffer to resuspend cells, sucking the supernatant by using the suction head after centrifuging at 300g, adding 200 mu L of electrotransfer buffer to resuspend the cells, respectively adding 3 mu g of pT2-HER2-synNotch-Cre-CAR plasmid and 3 mu g of pT2-EGFR vIII-synNotch-Cre-CAR plasmid into two parts of electrotransfer buffer resuspended T cells, respectively adding 2 mu g of transposase expression vector pCMV (or) -T7-SB100 into the cells, fully mixing the cells, adding the cells into an electrotransfer with the specification of 2mm, placing the cells into an electrotransfer, selecting human T lymphocyte electrotransfer sequence to perform electrotransfer, quickly adding the cells into a bath temperature to obtain a T cell culture medium, transferring the T cell culture medium into a culture disc after electrotransfer, and adjusting the concentration to 2 × 10⁶Each cell/mL, put into a cell incubator to be cultured.

2. HER2-synNotch-Cre-CAR-T, EGFR vIII-synNotch-Cre-CAR-T cell transduction efficiency assay

After 48 hours of culture, 1.0 × 10 was sampled⁶After each transduction of T cells, incubated with 1. mu.g/mL FITC-anti-strep tagII antibody at room temperature for 30 minutes, washed twice with physiological saline, FITC fluorescence signal was detected by flow cytometry, and the FITC positive cell ratio was measured, reflecting the ratio of CAR-T cells in total cells. Results of the HER2-synNotch-Cre-CAR-T, EGFRvIII-synNotch-Cre-CAR-T cell transfection efficiency test are respectively shown in FIG. 7. FIG. 7 shows that HER2-synNotch-Cre-CAR-T, EGFR vIII-synNotch-Cre-CAR-T cells were successfully prepared.

Example 4 preparation of Switch-oHSV-1-X oncolytic Virus

1. Construction of shuttle vector:

the oHSV-1-X virus is based on an HSV-117 strain, two copies of ICP34.5 and a single copy of ICP47(ICP34.5 and ICP47 are non-essential genes for virus replication) are deleted, and a CMV-EGFP-polyA tag gene expression frame is inserted into an ICP34.5 genomic site. The transformation of the oncolytic virus Switch-oHSV-1-X is carried out based on the oncolytic virus oHSV-1-X. In order to achieve the purposes of replacing an ICP4 gene promoter and adding Loxp-mCherry-polyA-Loxp (LSL), the genes are modified and synthesized by utilizing a homologous recombination method. Firstly, using DNAzol reagent to crack oHSV-1-X virus particles, and using an ethanol precipitation method to extract the genome DNA of the oHSV-1-X virus particles. The ICP4 gene is amplified by taking oHSV01-X DNA as a template in a segmented mode, and 1500bp upstream of an initiation codon and 1500bp downstream of a termination codon TAA of the ICP4 gene at two positions of a virus genome are amplified simultaneously to serve as two pairs of homology arms. Two pairs of homologous tails are sequentially connected together in an overlapping PCR mode according to the front and back sequence, single restriction endonucleases ClaI and XhoI are inserted between the front and back homologous arms, and single restriction endonucleases XbaI and HandIII are respectively inserted at the front and back ends. Then, the two pairs of homologous arms and the vector pUC57 are subjected to double enzyme digestion by XbaI and HandIII, the two pairs of homologous arms are respectively constructed into the vector pUC57 by using T4 ligase, and the vectors pUC57-TYW-1 and pUC57-TYW-2 are obtained through transformation and sequencing.

Artificially synthesizing a Loxp-mCherry-polyA-Loxp (LSL) gene segment, and obtaining a CMV promoter from a pcDNA3.1 vector by a PCR method. CMV, Loxp-mCherry-polyA-Loxp and ICP4 are fused into one by overlapping PCR means, ClaI and XhoI enzyme cutting sites are respectively added at two ends, and a CMV-LSL-ICP4 fragment is obtained. The CMV-LSL-ICP4 fragment and pUC57-TYW-1, pUC57-TYW-2 vectors were digested with ClaI and XhoI, and CMV-LSL-ICP4 was constructed into pUC57-T-YW-1 and pUC57-TYW-2 vectors by T4 ligase. Homologous recombination vectors pUC57-CMV-LSL-ICP4-1 and pUC57-CMV-LSL-ICP4-2 were obtained by sequencing. A large amount of plasmids are obtained through bacteria shaking and purification, and CMV-LSL-ICP4-1 and CMV-LSL-ICP4-2 fragments for virus genome recombination are obtained after double enzyme digestion, purification and recovery of XbaI and HandIII.

2. And (3) carrying out homologous recombination and screening to obtain a target virus:

Vero-ICP4 cells were transferred into a 100mm cell culture dish, and when the cell confluence reached about 85%, the O HSV01-X virus genomic DNA was co-transfected with linearized recombinant fragments CMV-LSL-ICP4-1 and CMV-LSL-ICP4-2 using a lipofectin to transfect Vero-ICP4 cells. Meanwhile, 2% of low-melting-point agarose is sterilized at high temperature, and is placed into a sealed container after being sterilized and is placed into a 56 ℃ oven for later use to prevent cooling and solidification; 2 XDMEM medium was prepared with DMEM powder and filter sterilized for use. After 6 to 8 hours of lipofection of Vero cells, the medium was discarded and the cells were mixed with 2 × DMEM medium and 2% low melting agarose 1:1, mixing uniformly, quickly covering Vero cells of cotransfection virus genome and recombinant fragment when the Vero cells are cooled to about 37 ℃, then putting the Vero cells into a refrigerator at 4 ℃ for about 20 minutes to solidify, and then putting the Vero cells back into the incubator to continue culturing. After 2 to 4 days cytolytic plaques appeared upon homologous recombination and packaging of the virus, there was a single viral genome in which ICP4 was replaced by the CMV-LSL-ICP4 fragment, which caused the recombined virus to emit red lytic plaques. At this time, cells with a plurality of red lysis spots are picked up by a suction head and placed into 200 mu L of culture medium for repeated freeze thawing for 3 times to release virus particles, vero cells with the cell density of about 90 percent are infected again, the culture is continued by covering the agarose culture medium by the same method after three hours, and the red spots are picked up when cell lysis plaques appear after 2 to 4 days. And finally obtaining a virus strain with very high purity through 6-8 repeated virus selection and purification, and obtaining the virus strain with very high purity through sequencing and screening, wherein ICP4 genes in the genome of the oHSV-1-X virus are all replaced by CMV-LSL-ICP4, namely the controllable oncolytic virus Switch-oHSV-1-X with the Switch. The scheme for constructing Switch-oHSV-1-X is shown in figure 6.

Example 5 synNotch-Cre-CAR-T cell load oncolytic virus Switch-oHSV-1-X ratio assay of oncolytic virus Switch-oHSV-1-X

1. synNotch-Cre-CAR-T cells were loaded with oncolytic virus Switch-oHSV-1-X:

count respectively 1 × 10⁷HER2-synNotch-Cre-CAR-T and EGFR vIII-synNotch-Cre-CAR-T cells, centrifuging at 300g to remove culture medium, adding 1mL of T cell culture medium to adjust cell concentration to 1 × 10⁷one/mL. Adding Switch-oHSV-1-X according to MOI (1) and Polybrene with the final concentration of 5 mu g/mL, fully mixing, placing at 37 ℃ and 5% CO₂Transfecting in an incubator for 3 hours, centrifuging for 5 minutes at 300g after 3 hours to discard the medium containing the virus suspension, suspending the cells by adding 5mL of T cell culture medium, centrifuging for 5 minutes at 300g to discard the medium, repeating the above washing steps for 2 to 3 times, and adjusting the cell density to 2 × 10⁶Put the mixture in a container at 37 ℃ with 5% CO₂Culturing in an incubator for later use.

2. synNotch-Cre-CAR-T cell proportion detection loaded with oncolytic virus Switch-oHSV-1-X:

HER2-synNotch-Cre-CAR-T and EGFR vIII-synNotch-Cre-CAR-T cells are placed in an incubator to be cultured for 48 hours to 72 hours, cell green fluorescent protein EGFP of the loaded viruses can be expressed, the proportion of the EGFP is detected by the number of flow cells, the proportion of the HER2-synNotch-Cre-CAR-T and EGFR vIII-synNotch-Cre-CAR-T loaded oncolytic viruses can be determined, and the flow detection result is shown in figure 8, and shows that HER2-synNotch-Cre-CAR-T and EGFR vIII-synNotch-Cre-T cells are successfully loaded with the oncolytic virus Switch-oCAR-1-X.

Example 6 synNotch-Cre-CAR-T conditionally initiates detection of Switch-oHSV-1-X:

1. Switch-oHSV-1-X loaded synNotch-Cre-CAR-T receives target cell stimulation:

respectively loading the Switch-oHSV-1-X virus to HER2-synNotch-Cre-CAR-T and EGFR vIII-synNotch-Cre-CAR-T cells, then respectively incubating HER2-synNotch-Cre-CAR-T and corresponding target cells MCF-7(HER2+), EGFR vIII-synNotch-Cre-CAR-T and corresponding target cells U251 MG (EGFR vIII +) for 3 hours according to an effective target ratio of 2: 1, then lightly blowing and suspending HER2-synNotch-Cre-CAR-T and EGFR vIII-synNotch-Cre-T cells (target cells are in an adherent state) by using a suction head, centrifuging the culture medium for 5 minutes by sucking the culture medium into a 15mL centrifuge tube, discarding the culture medium, and adjusting the density to 2 × 10 by using the culture medium basic suspension of T cells⁶Perml of CO₂The incubator continues to culture. After 36 hours, the same number of HER2-synNotch-Cre-CAR-T cells, EGFR vIII-synNotch-Cre-CAR-T cells, and HER2-synNotch-Cre-CAR-T and EGFR vIII-synNotch-Cre-CAR-T cells which were not stimulated by target cells were counted, and the genome was extracted by the DNAzol method.

2. Detecting the genome copy number of oncolytic virus Switch-oHSV-1-X in synNotch-Cre-CAR-T cells by using a qPCR method:

the specific method for detecting the copy number of the genome of oncolytic virus Switch-oHSV-1-X in synNotch-Cre-CAR-T cells comprises the steps of selecting a conserved sequence of the genome of Switch-oHSV-1-X as a target sequence for amplifying oncolytic virus Switch-oHSV-1-X, using human β -globin gene as an internal reference gene, amplifying a target gene and the internal reference gene in the same reaction system, marking different fluorescent groups by the target gene and the internal reference gene, designing a specific primer probe on the target gene, using a plasmid with the target gene as a standard substance, and using the standard substance with known initial copy number to prepare a standard kojiThe copy number of β -globin of the sample to be detected can be obtained by the same principle, half of the copy number of β -globin is the cell number of the sample to be detected, namely β -globin copy number is used for calibrating the cell number in the sample to be detected, the copy number of the Switch-oHSV-1-X oncolytic virus relative to the number of sync-Cre-CAR-T cells can be expressed as 2 × target gene copy number/β -globin gene copy number, the experimental result is shown in figure 9, and the result shows that compared with a control group without target cells, HER 2-sync-Cre-CAR-T stimulated by the target cells detects about 8000copies/10⁶About 6000copies/10 were detected in cells' Switch-oHSV-1-X oncolytic virus, EGFR vIII-synNotch-Cre-CAR-T cells⁶Switch-oHSV-1-X oncolytic virus by cells, indicating that HER2-synNotch-Cre-CAR-T cells and EGFR vIII-synNotch-Cre-CAR-T cells can successfully turn on Switch-oHSV-1-X replication.

Example 7 synNotch-Cre-CAR-T specifically presents Switch-oHSV-1-X to target cells:

1. Synnotch-Cre-CAR-T cell loaded with Switch-oHSV-1-X oncolytic virus and target cell are incubated together

Construction of HER2-CAR-T cells and EGFRvIII-CAR-T cells without synNotch-Cre system HER2-CAR-T cells and EGFRvIII-CAR-T cells to which oncolytic virus Switch-oHSV-1-X was added were treated in the same manner as synNotch-Cre-CAR-T cells were loaded with oncolytic virus Switch-oHSV-1-X in example 5. HER2-CAR-T cells, Switch-oHSV-1-X virus-loaded HER2-synNotch-Cre-CAR-T, and its target cell MCF-7 and negative control target cell K562 were compared in an effective to target ratio of 2: 1 co-incubation. Similarly, EGFRvIII-CAR-T cells, EGFR vIII-synNotch-Cre-CAR-T cells loaded with Switch-oHSV-1-X virus, and their target cells U251-MG and negative control target cell K562 were mixed in an effective to target ratio of 2: 1 co-incubation. After 12 to 24 hours, the cells of HER2-synNotch-Cre-CAR-T and EGFR vIII-synNotch-Cre-CAR-T were gently blown down with a suction head, the target cells (which are in an adherent state) were not blown down from the dish, the suspension medium and the T cells therein were aspirated, and the adherent target cells were washed three times with PBS, and the T cells were washed clean as much as possible. The target cells were then digested from the bottom of the dish by adding a small amount of trypsin, quenched with the appropriate amount of media and transferred to a 1.5mL centrifuge tube. The same number of target cells and negative control target cells were used to extract their genomic DNA by DNAzol method.

1.2, detecting the copy number of the genome of the oncolytic virus Switch-oHSV-1-X in a target cell by using a qPCR method:

the copy number of the genome of the oncolytic virus Switch-oHSV-1-X and the copy number of beta-globin of target cells MCF-7 and U251-MG are detected by the method in example 6, and the copy number of the oncolytic virus Switch-oHSV-1-X relative to the number of the target cells can be expressed as follows: 2 Xthe copy number of the target gene/copy number of. beta. -globin. The results are shown in FIG. 10, and show that after the synNotch-Cre-CAR-T cell loaded with the Switch-oHSV-1-X oncolytic virus is incubated with the target cell, a large amount of the Switch-oHSV-1-X oncolytic virus is detected in the target cell, and the synNotch-Cre-CAR-T can present the oncolytic disease Switch-oHSV-1-X to the target cell and can rapidly replicate in the target cell.

Furthermore, the data for the HER2-CAR-T + Switch-oHSV-1-X and EGFRvIII-CAR-T + Switch-oHSV-1-X groups indicate that Switch-oHSV-1-X cannot be presented to target cells without the synNotch-Cre element and cannot turn on replication of oncolytic-oHSV-1-X.

Example 8 synNotch-Cre-CAR-T kills target cells synergistically with Switch-oHSV-1-X:

to examine the universality of synNotch-Cre-CAR-T presenting oncolytic virus Switch-oHSV-1-X in solid tumor combination applications, we selected two targets, HER2 and EGFR vIII, where HER2-synNotch-Cre-CAR-T corresponds to target cells MCF-7(HER2+), and EGFRvIII-synNotch-Cre-CAR-T corresponds to target cells U251-MG (EGFRvIII +).

1. Construction of Luciferase target cell stable cell line

In the scheme, target cells are marked by adopting a mode of constructing a stable cell line by using lentiviruses, and firstly, 2 × 10 is respectively added⁵Transferring the MCF-7 and U251-MG cell lines into a T25 culture flask and adding CO₂The cells are cultured in an incubator overnight, and when the confluence degree of the cells is about 75 percent, the cells are transferred into PTK-Luci-Puro lentivirus (the lentivirus carries the silver fire worm Luciferase gene and the resistance gene Puromycin) according to the MOI of 3. After transfection for about 4 hoursAnd (3) sucking the virus-containing culture medium supernatant by using the suction head, slowly adding fresh DMEM complete culture medium, and putting the DMEM complete culture medium into the cell culture box for continuous culture. When the cells grow to be full of the bottom of the dish, the cells are passaged according to the proportion of 1/3. Puromycin was added at a working concentration of 2. mu.g/mL for selection 72 hours after lentivirus transfer, during which time cell confluency continued to 1/3 passages and the working concentration of Puromycin was maintained at 2. mu.g/mL throughout. The MCF-7 and U251-MG cells with the purity of more than 98 percent and capable of stably transforming the Luciferase, namely MCF-7-Luci and U251-MG-Luci, can be obtained after continuous screening for about 7 to 10 days.

2. synNotch-Cre-CAR-T target cell killing experiments loaded with oncolytic virus Switch-oHSV-1-X:

taking the target cell MCF-7-Luci according to the weight ratio of 1:1 and 4:1, respectively adding HER2-synNotch-Cre-CAR-T cells loaded with Switch-oHSV-1-X; similarly, in the U251-MG-Luci target cell corresponding to EGFR vIII target, the ratio of 1:1 and 4:1, respectively adding EGFRvIII-synNotch-Cre-CAR-T cells loaded with Switch-oHSV-1-X. In addition, an equal volume of PBS is added as a negative control group, an equal volume of cell lysate is added as a positive control group, and then the cells are placed into a cell culture box for incubation for 6-8 days. After the incubation was complete the luciferase substrate D (-) -Luciferin was added. The fluorescence intensity was measured with a microplate reader, and the detection time per well was 1000 ms. And after the detection is finished, counting the fluorescence intensity K value of each sample. The fluorescence intensity of the PBS-added negative control group was recorded as K_CThe killing effect is 0%, the cell lysate group is used as a positive control of the death of all cells by lysis, namely K_M. The killing efficiency of each group was calculated as follows:

killing efficiency%_C-K)/(K_C-K_M) × 100 percent 100% lysis of target cells

The results show (see fig. 11), at 1:1, HER2-synNotch-Cre-CAR-T cells loaded with oncolytic virus Switch-oHSV-1-X had a killing efficiency of about 51% against target cell MCF-7, significantly higher than HER2-synNotch-Cre-CAR-T cell group alone (about 10%) and Switch-oHSV-1-X oncolytic virus group alone (about 1%), again at an effective to target ratio of 4:1, the killing efficiency of the HER2-synNotch-Cre-CAR-T cell group loaded with the Switch-oHSV-1-X is about 84 percent, which is remarkably higher than that of the HER2-synNotch-Cre-CAR-T cell group (about 17 percent) and that of the Switch-oHSV-1-X oncolytic virus group (about 6 percent), and the results show that the HER2-synNotch-Cre-CAR-T cells loaded with the Switch-oHSV-1-X can obviously promote the lysis of HER + target cells MCF-7-Luci compared with other two groups.

Similarly, the EGFR vIII-synNotch-Cre-CAR-T cell loaded with the oncolytic virus Switch-oHSV-1-X can also obviously promote the lysis of the EGFR vIII target cell U251-MG-Luci (the tumor killing efficiency is about 62 percent and 91 percent) when the effective target ratio is 1:1 and 4:1, and the tumor killing effect is obviously better than that of the EGFR vIII-synNotch-Cre-CAR-T cell and the oncolytic virus Switch-oHSV-1-X alone.

In addition, the data of the Switch-oHSV-1-X group show that the oncolytic virus added with the LSL starting Switch has safety before being started without Cre and hardly has the capability of killing cells; the synNotch-Cre original has also been shown to play an essential role in turning on oncolytic virus replication.

The above examples and experimental data show that synNotch-Cre-CAR-T can conditionally activate the replication of Switch-oHSV-1-X in cells, and presents to adjacent target cells, and together generate synergistic effect of killing target cells. Solves the drug administration problem of oncolytic virus and relieves the obvious defects of CAR-T in the treatment of solid tumors, and can generate the synergistic anti-tumor effect with CAR-T immunotherapy, thereby providing a new treatment scheme for the solid tumors.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Sequence listing

<110> Wuhan Borui Rui Da Biotech Co., Ltd

<120> CAR-T technology for presenting and activating HSV-1 type oncolytic virus and application thereof

<130>1

<160>32

<170>PatentIn version 3.5

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<213>Artificial Sequence

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<223> Signal peptide

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210>2

<211>326

<212>PRT

<213>Artificial Sequence

<220>

<223> Notch Core amino acid sequence

<400>2

Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro Pro

1 5 10 15

Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp Ala

20 25 30

Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly Trp

35 40 45

Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn Cys

50 55 60

Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys Asp

65 70 75 80

Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys Gln

85 90 95

Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys Asp

100 105 110

His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu Cys

115 120 125

Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu Ala

130135 140

Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu Arg

145 150 155 160

Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His Thr

165 170 175

Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe Pro

180 185 190

Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ser

195 200 205

Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly Gly

210 215 220

Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile Val

225 230 235 240

Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln Cys

245 250 255

Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser

260 265 270

Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser Glu

275 280 285

Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala

290 295300

Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu

305 310 315 320

Ser Arg Lys Arg Arg Arg

325

<210>3

<211>343

<212>PRT

<213>Artificial Sequence

<220>

<223> Cre recombinase amino acid sequence

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Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val

1 5 10 15

Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe Arg

20 25 30

Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser Val

35 40 45

Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys Trp Phe

50 55 60

Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln Ala

65 70 75 80

Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln Leu Asn

85 90 95

Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser Asn Ala

100 105 110

Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp Ala Gly

115 120 125

Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe Asp Gln

130 135 140

Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile Arg Asn

145 150 155 160

Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala Glu

165 170 175

Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly Arg

180 185 190

Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala Gly

195 200 205

Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg Trp

210 215 220

Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cys

225 230 235 240

Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser Gln Leu

245 250 255

Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg Leu Ile

260 265 270

Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu Ala Trp Ser Gly

275 280 285

His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala Gly Val

290 295 300

Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val Asn Ile

305 310 315 320

Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr Gly Ala Met Val

325 330 335

Arg Leu Leu Glu Asp Gly Asp

340

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<212>PRT

<213>Artificial Sequence

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<223> CD8Hinge amino acid sequence

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Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210>5

<211>68

<212>PRT

<213>Artificial Sequence

<220>

<223> CD28TM + ICD amino acid sequence

<400>5

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser

20 25 30

Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly

35 40 45

Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala

50 55 60

Ala Tyr Arg Ser

65

<210>6

<211>42

<212>PRT

<213>Artificial Sequence

<220>

<223>4-1BB amino acid sequence

<400>6

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 510 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

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<211>112

<212>PRT

<213>Artificial Sequence

<220>

<223> CD3 ζ amino acid sequence

<400>7

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

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<212>PRT

<213>Artificial Sequence

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<223> mCherry amino acid sequence

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Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe

1 5 10 15

Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe

20 25 30

Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr

35 40 45

Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp

50 55 60

Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His

65 70 75 80

Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe

85 90 95

Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val

100 105110

Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys

115 120 125

Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys

130 135 140

Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly

145 150 155 160

Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly

165 170 175

His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val

180 185 190

Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser

195 200 205

His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly

210 215 220

Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210>9

<211>122

<212>DNA

<213>Artificial Sequence

<220>

<223> SV40polyA nucleotide sequence

<400>9

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120

ta 122

<210>10

<211>34

<212>DNA

<213>Artificial Sequence

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<223> Forward Loxp nucleotide sequence

<400>10

ataacttcgt atagcataca ttatacgaag ttat 34

<210>11

<211>116

<212>PRT

<213>Artificial Sequence

<220>

<223> EGFR vIII-VH amino acid sequence

<400>11

Arg Pro Glu Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

1 5 10 15

Gly Glu Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Phe Asn Ile Glu

20 25 30

Asp Tyr Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

35 40 45

Trp Met Gly Arg Ile Asp Pro Glu Asn Asp Glu Thr Lys Tyr Gly Pro

50 55 60

Ile Phe Gln Gly His Val Thr Ile Ser Ala Asp Thr Ser Ile Asn Thr

65 70 75 80

Val Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Ala Phe Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210>12

<211>112

<212>PRT

<213>Artificial Sequence

<220>

<223> EGFR vIII-VL amino acid sequence

<400>12

Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Arg Leu Ile Ser Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuThr Ile

65 70 75 80

Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Gly Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>13

<211>119

<212>PRT

<213>Artificial Sequence

<220>

<223> HER2-VH amino acid sequence

<400>13

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Pro Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Ser Thr Gly Glu Ser Thr Phe Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Asp Phe Ser Leu Glu Thr Ser Ala Asn Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys SerGlu Asp Met Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Trp Glu Val Tyr His Gly Tyr Val Pro Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210>14

<211>107

<212>PRT

<213>Artificial Sequence

<220>

<223> HER2-VL amino acid sequence

<400>14

Asp Ile Gln Leu Thr Gln Ser His Lys Phe Leu Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Tyr Asn Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Ser Arg Tyr Thr Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Pro Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln His Phe Arg Thr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210>15

<211>15

<212>PRT

<213>Artificial Sequence

<220>

<223> Linker amino acid sequence

<400>15

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>16

<211>19

<212>PRT

<213>Artificial Sequence

<220>

<223> P2A peptide amino acid sequence

<400>16

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210>17

<211>63

<212>DNA

<213>Artificial Sequence

<220>

<223> CD8 alpha signal peptide nucleotide sequence

<400>17

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210>18

<211>357

<212>DNA

<213>Artificial Sequence

<220>

<223> HER2-VH nucleotide sequence

<400>18

caggtgcagc tgcagcagag cggccccgag ctgaaaaagc ccggcgagac agtgaagatc 60

agctgcaaag ccagcggcta ccccttcaca aactacggca tgaactgggt gaagcaggcc 120

cccggccagg gactgaaatg gatgggctgg atcaacacca gcaccggcga gagcaccttc 180

gccgacgact tcaaaggcag attcgacttc agcctggaga ccagcgctaa cacagcctac 240

ctgcagatta acaacctgaa aagcgaagac atggccacct acttctgtgc ccgctgggag 300

gtgtatcacg gctacgtgcc ctactgggga cagggaacca ccgtgaccgt gtctagc 357

<210>19

<211>321

<212>DNA

<213>Artificial Sequence

<220>

<223> HER2-VL nucleotide sequence

<400>19

gacatccagc tgacacagag ccacaagttc ctgagcacca gcgtgggaga cagagtgtct 60

atcacctgca aggctagtca ggacgtgtat aacgccgtgg cctggtacca gcagaaaccc 120

ggccagagcc ccaagctgct gatctactcc gccagcagca gatacacagg cgtgcccagc 180

agatttacag gaagcggcag cggccccgac tttaccttca ctatcagcag cgtgcaggcc 240

gaggacctgg ccgtgtactt ctgccagcag cacttcagga ccccattcac cttcggaagc 300

ggcaccaagc tggagatcaa g 321

<210>20

<211>348

<212>DNA

<213>Artificial Sequence

<220>

<223> EGFR vIII-VH nucleotide sequence

<400>20

cggcccgaga ttcagctcgt gcaatcggga gcggaagtca agaagccagg agagtccttg 60

cggatctcat gcaagggtag cggctttaac atcgaggatt actacatcca ctgggtgagg 120

cagatgccgg ggaagggact cgaatggatg ggacggatcg acccagaaaa cgacgaaact 180

aagtacggtc cgatcttcca aggccatgtg actattagcg ccgatacttc aatcaatacc 240

gtgtatctgc aatggtcctc attgaaagcc tcagataccg cgatgtacta ctgtgctttc 300

agaggagggg tctactgggg acagggaact accgtgactg tctcgtcc 348

<210>21

<211>336

<212>DNA

<213>Artificial Sequence

<220>

<223> EGFR vIII-VL nucleotide sequence

<400>21

gacgtcgtga tgacccagag ccctgacagc ctggcagtga gcctgggcga aagagctacc 60

attaactgca aatcgtcgca gagcctgctg gactcggacg gaaaaacgta cctcaattgg 120

ctgcagcaaa agcctggcca gccaccgaag cgccttatct cactggtgtc gaagctggat 180

tcgggagtgc ccgatcgctt ctccggctcg ggatcgggta ctgacttcac cctcactatc 240

tcctcgcttc aagcagagga cgtggccgtc tactactgct ggcagggaac ccactttccg 300

ggaaccttcg gcggagggac gaaagtggag atcaag 336

<210>22

<211>45

<212>DNA

<213>Artificial Sequence

<220>

<223> Linker nucleotide sequence

<400>22

ggcggaggcg ggtcaggagg tggcggcagc ggaggaggag ggtcc 45

<210>23

<211>135

<212>DNA

<213>Artificial Sequence

<220>

<223> CD8Hinge nucleotide sequence

<400>23

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210>24

<211>204

<212>DNA

<213>Artificial Sequence

<220>

<223> CD28TM + ICD nucleotide sequence

<400>24

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 120

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 180

cgcgacttcg cagcctatcg ctcc 204

<210>25

<211>126

<212>DNA

<213>Artificial Sequence

<220>

<223>4-1BB nucleotide sequence

<400>25

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210>26

<211>336

<212>DNA

<213>Artificial Sequence

<220>

<223> CD3 zeta nucleotide sequence

<400>26

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210>27

<211>27

<212>DNA

<213>Artificial Sequence

<220>

<223> Strep tag II nucleotide sequence

<400>27

aactggagcc acccccagtt cgagaag 27

<210>28

<211>978

<212>DNA

<213>Artificial Sequence

<220>

<223> nucleotide sequence of Notch Core

<400>28

atcctggact acagcttcac aggtggcgct gggcgcgaca ttcccccacc gcagattgag 60

gaggcctgtg agctgcctga gtgccaggtg gatgcaggca ataaggtctg caacctgcag 120

tgtaataatc acgcatgtgg ctgggatggt ggcgactgct ccctcaactt caatgacccc 180

tggaagaact gcacgcagtc tctacagtgc tggaagtatt ttagcgacgg ccactgtgac 240

agccagtgca actcggccgg ctgcctcttt gatggcttcg actgccagct caccgaggga 300

cagtgcaacc ccctgtatga ccagtactgc aaggaccact tcagtgatgg ccactgcgac 360

cagggctgta acagtgccga atgtgagtgg gatggcctag actgtgctga gcatgtaccc 420

gagcggctgg cagccggcac cctggtgctg gtggtgctgc ttccacccga ccagctacgg 480

aacaactcct tccactttct gcgggagctc agccacgtgc tgcacaccaa cgtggtcttc 540

aagcgtgatg cgcaaggcca gcagatgatc ttcccgtact atggccacga ggaagagctg 600

cgcaagcacc caatcaagcg ctctacagtg ggttgggcca cctcttcact gcttcctggt 660

accagtggtg ggcgccagcg cagggagctg gaccccatgg acatccgtgg ctccattgtc 720

tacctggaga tcgacaaccg gcaatgtgtg cagtcatcct cgcagtgctt ccagagtgcc 780

accgatgtgg ctgccttcct aggtgctctt gcgtcacttg gcagcctcaa tattccttac 840

aagattgagg ccgtgaagag tgagccggtg gagcctccgc tgccctcgca gctgcacctc 900

atgtacgtgg cagcggccgc cttcgtgctc ctgttctttg tgggctgtgg ggtgctgctg 960

tcccgcaagc gccggcgg 978

<210>29

<211>1029

<212>DNA

<213>Artificial Sequence

<220>

<223> Cre recombinase nucleotide sequence

<400>29

atgtccaatt tactgaccgt acaccaaaat ttgcctgcat taccggtcga tgcaacgagt 60

gatgaggttc gcaagaacct gatggacatg ttcagggatc gccaggcgtt ttctgagcat 120

acctggaaaa tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac 180

cggaaatggt ttcccgcaga acctgaagat gttcgcgatt atcttctata tcttcaggcg 240

cgcggtctgg cagtaaaaac tatccagcaa catttgggcc agctaaacat gcttcatcgt 300

cggtccgggc tgccacgacc aagtgacagc aatgctgttt cactggttat gcggcggatc 360

cgaaaagaaa acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact 420

gatttcgacc aggttcgttc actcatggaa aatagcgatc gctgccagga tatacgtaat 480

ctggcatttc tggggattgc ttataacacc ctgttacgta tagccgaaat tgccaggatc 540

agggttaaag atatctcacg tactgacggt gggagaatgt taatccatat tggcagaacg 600

aaaacgctgg ttagcaccgc aggtgtagag aaggcactta gcctgggggt aactaaactg 660

gtcgagcgat ggatttccgt ctctggtgta gctgatgatc cgaataacta cctgttttgc 720

cgggtcagaa aaaatggtgt tgccgcgcca tctgccacca gccagctatc aactcgcgcc 780

ctggaaggga tttttgaagc aactcatcga ttgatttacg gcgctaagga tgactctggt 840

cagagatacc tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg agatatggcc 900

cgcgctggag tttcaatacc ggagatcatg caagctggtg gctggaccaa tgtaaatatt 960

gtcatgaact atatccgtaa cctggatagt gaaacagggg caatggtgcg cctgctggaa 1020

gatggcgat 1029

<210>30

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223> Strep tag II amino acid sequence

<400>30

Asn Trp Ser His Pro Gln Phe Glu Lys

1 5

<210>31

<211>14

<212>PRT

<213>Artificial Sequence

<220>

<223> Flag amino acid sequence

<400>31

Asn Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser

1 5 10

<210>32

<211>42

<212>DNA

<213>Artificial Sequence

<220>

<223> Flag nucleotide sequence

<400>32

aactggagcc acccccagtt cgagaagggc ggtggcggaa gc 42

Claims

1. A system for killing solid tumor cells, which comprises HSV-1 type oncolytic virus, a chimeric antigen receptor and an scFv-synNotch-Cre element; wherein scFv is a single-chain antibody targeting tumor specific antigen, Notch is a core element of transmembrane and splicing site of Notch, and Cre is recombinase based on Loxp site.

2. The system of claim 1, wherein the HSV-1-type oncolytic virus lacks an ICP34.5 gene and an ICP47 gene, and a CMV promoter and a Loxp-Stop-Loxp sequence are inserted by pre-homologous recombination of the ICP4 gene.

3. The system of claim 2, wherein the Stop is formed by sequentially connecting a CDS region of a gene and a transcription termination signal; the CDS region of the gene is a fluorescent protein gene, the nucleotide sequence of the transcription termination signal is shown as SEQ ID NO.9, and the nucleotide sequence of Loxp is shown as SEQ ID NO. 10.

4. The system of claim 3, wherein the amino acid sequence of the fluorescent protein is set forth in SEQ ID No. 8.

5. The system of any one of claims 1-4, wherein the scFv-synNotch-Cre element comprises a signal peptide, scFv, Flag, Notch Core, and Cre recombinase.

6. The system of claim 5, wherein the amino acid sequence of the signal peptide is as set forth in SEQ ID No. 1; the scFv is a specific protein capable of specifically recognizing the surface of the solid tumor; the amino acid sequence of the Notch Core is shown as SEQ ID NO. 2; the amino acid sequence of the Cre recombinase is shown in SEQ ID NO. 3.

7. The system of claim 6, wherein the chimeric antigen receptor comprises a signal peptide, ScFv, Strep tag ii, CD8hinge, CD28TM + ICD, 4-1BB, and CD3 ζ; the amino acid sequence of the signal peptide is shown as SEQ ID NO. 1; the Strep tag II amino acid sequence is shown as SEQ ID NO. 30; the scFv is a specific protein capable of specifically recognizing the surface of the solid tumor; the amino acid sequence of the CD8hinge is shown as SEQ ID NO. 4; the amino acid sequence of the CD28TM + ICD is shown in SEQ ID NO. 5; the amino acid sequence of the 4-1BB is shown as SEQ ID NO. 6; the amino acid sequence of the CD3 zeta is shown in SEQ ID NO. 7.

8. The system of claim 6 or 7, wherein the scFv is EGFR vIII or/and HER 2; the scFv is composed of a heavy chain variable region V_HLinker connecting polypeptide and light chain variable region V_LComposition is carried out; wherein the amino acid sequence of the variable region VH of the EGFR vIII-scFv is shown in SEQ ID NO.11, and the amino acid sequence of the variable region VL of the EGFR vIII-scFv is shown in SEQ ID NO. 12; the amino acid sequence of the variable region VH of HER2-scFv is shown in SEQ ID NO.13, and the HER2-scFv light chainThe amino acid sequence of the variable region VL is shown as SEQ ID NO. 14; the amino acid sequence of the connecting polypeptide Linker is shown as SEQ ID NO. 15.

9. An immune cell comprising the expression system of any one of claims 1-4 and 6-7.

10. Use of a system according to any one of claims 1-4 and 6-7 for the preparation of a medicament for the prevention or/and treatment of tumors.